Discovery of gefitinib-1,2,3-triazole derivatives against lung cancer via inducing apoptosis and inhibiting the colony formation

A series of 20 novel gefitinib derivatives incorporating the 1,2,3-triazole moiety were designed and synthesized. The synthesized compounds were evaluated for their potential anticancer activity against EGFR wild-type human non-small cell lung cancer cells (NCI-H1299, A549) and human lung adenocarcinoma cells (NCI-H1437) as non-small cell lung cancer. In comparison to gefitinib, Initial biological assessments revealed that several compounds exhibited potent anti-proliferative activity against these cancer cell lines. Notably, compounds 7a and 7j demonstrated the most pronounced effects, with an IC50 value of 3.94 ± 0.17 µmol L−1 (NCI-H1299), 3.16 ± 0.11 µmol L−1 (A549), and 1.83 ± 0.13 µmol L−1 (NCI-H1437) for 7a, and an IC50 value of 3.84 ± 0.22 µmol L−1 (NCI-H1299), 3.86 ± 0.38 µmol L−1 (A549), and 1.69 ± 0.25 µmol L−1 (NCI-H1437) for 7j. These two compounds could inhibit the colony formation and migration ability of H1299 cells, and induce apoptosis in H1299 cells. Acute toxicity experiments on mice demonstrated that compound 7a exhibited low toxicity in mice. Based on these results, it is proposed that 7a and 7j could potentially be developed as novel drugs for the treatment of lung cancer.

www.nature.com/scientificreports/EGFR-TKIs exhibit a robust inhibitory effect on EGFR mutant lung cancer cells; however, their inhibitory activity against wild-type lung cancer cells is comparatively weaker.The objective of this study is to explore structural modifications of EGFR-TKIs in order to augment their inhibitory activity specifically against wildtype lung cancer cells.
The 1,2,3-triazole moiety a nitrogenous heterocyclic compound consisting of a five-membered ring.It has found widespread use in various fields such as medicine, pesticides, and more, owing to its ability to serve as a bioisosteric replacement for diverse structures including acyl groups, carboxylic acid groups, and heterocyclic groups.These compounds can bind to tumor cells through various non-covalent interactions such as hydrogen bonding, van der Waals forces, and metal chelation.They induce tumor cell apoptosis through multiple mechanisms, including blocking the growth cycle, inhibiting cell proliferation, and reducing mitochondrial transmembrane potential.These compounds have potential anti-tumor activity.
In summary, the integration of 1,2,3-triazole with other pharmacophores represents a highly effective approach in the development of novel anti-cancer drugs (Fig. 2, 6) 19 .The diverse studies presented demonstrate the remarkable potential of 1,2,3-triazole-modified compounds in cancer treatment, offering insights into their mechanisms and promising outcomes in cell-based studies.
To identify compounds with improved inhibitory effects on wild-type lung cancer cells, we modified gefitinib using a click reaction, introducing a 1,2,3-triazole moiety into its structure.We hoped that this modification would exhibit inhibitory effects on wild-type lung cancer cells.Using gefitinib as a positive control, we employed the CCK-8 assay to investigate the in vitro anti-tumor activity on wild-type human non-small cell lung cancer cells (NCI-H1299, A549) and human lung adenocarcinoma cells (NCI-1437).Furthermore, we studied the impact on tumor cell colony formation, migration ability, and apoptosis.

Chemistry
The synthetic strategy for the preparation of the target molecules is illustrated through a literature review in Scheme 1 [20][21][22] .Nitration of 1 was realized using HNO 3 /H 2 SO 4 as the nitration agent, and the resulted compound 2 was readily converted to compound 3 under the conventional hydrogenation conditions.Cyclization reaction between 3 and formamide produced compound 4. The chlorination of hydroxyl group with phosphorus oxychloride produced compound 5. Compound 6 was obtained after reaction of 5 and 3-aminophenylacetylene.Finally, 1,2,3-triazole gefitinib derivatives 7a-7t was synthesized through a copper(I)-catalyzed azide-alkyne cycloaddition reaction.The reaction conditions of these operations were mild and easy to control.The structures of the key www.nature.com/scientificreports/intermediates and all target compounds were confirmed through nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR) and high-resolution mass spectrometry (HRMS).The detail and figures of NMR spectrums and HRMS were included in the supplementary files.

Compounds 7a-7t suppressed cancer cells viability
In this study, the CCK-8 assay was employed to investigate the cell inhibitory effects of compounds 7a-7t on human lung cancer cells NCI-H1299, NCI-H1437, and A549, with gefitinib used as a positive control.The results (Table 1) indicated that almost all compounds exhibited inhibitory effects on NCI-H1299, NCI-H1437, and A549 lung cancer cells, with compounds 7a and 7j showing the best performance.Specifically, in the NCI-H1299 cell line, the IC 50 values for 7a and 7j were 3.94 ± 0.17 μM and 3.84 ± 0.22 μM, respectively.In the NCI-H1437 cell line, the IC 50 values for 7a and 7j were 1.83 ± 0.13 μM and 1.69 ± 0.25 μM, respectively.In the A549 cell line, the IC 50 values for 7a and 7j were 3.16 ± 0.11 μM and 3.86 ± 0.38 μM, respectively.These values were all superior to the inhibitory effects of gefitinib in the three cell lines (IC 50 = 14.62 ± 0.90 μM, IC 50 = 20.56 ± 2.45 μM, and IC 50 = 14.62 ± 0.43 μM, respectively).The experimental results suggest that the gefitinib derivatives have higher cytotoxicity and play a pro-apoptotic role in lung cancer cells.Compounds 7a and 7j exhibited IC 50 values of 18.87 + 1.03 μM and 17.68 ± 0.52 μM respectively in the L02 cell line.Moreover, our study demonstrated that these compounds are less toxic to normal hepatocytes and more effective in killing lung cancer cells, with a survival rate of normal hepatocytes L02 treated with 7a and 7j at a concentration of 4 μM between 70% and 90%.These findings suggest that compounds 7a and 7j hold great potential as clinical therapeutic agents for lung cancer (Table 2).The introduction of phenyl or benzyl groups had a notable impact on the activity of the compounds.Notably, when the triazole linkage was benzyl, the activity was superior to phenyl.The study clearly demonstrates the successful structural modification of gefitinib, resulting in significant differences in compound activity.Furthermore, the addition of bromine atoms in the neighbouring position of the benzyl ring or the addition of fluorine or chlorine atoms in the interstitial position significantly improved the in vitro antitumour activity of the compounds.www.nature.com/scientificreports/

Compounds 7a and 7j induce apoptosis in H1299 cells
To elucidate whether the inhibitory effects on cell proliferation and cytotoxicity of these compounds are associated with apoptosis, we conducted relevant experiments focusing on compounds 7a and 7j, which demonstrated good inhibitory effects on the proliferation of the three types of cancer cells.H1299 cells were treated with different concentrations of 7a and 7j for 48 h.The cells were stained with Annexin-V and PI, and the proportion of apoptotic cells was detected using flow cytometry.
As shown in Fig. 3A, after treatment with compound 7a, the total apoptotic cell proportions in H1299 were 19.03 ± 2.10% (2 μmol/L), 28.73 ± 1.12% (4 μmol/L), and 50.1 ± 2.91% (8 μmol/L) (Fig. 3).Following treatment with compound 7j, the total apoptotic cell proportions in H1299 were 14.97 ± 1.54% (2 μmol/L), 27.07 ± 2.77% (4 μmol/L), and 65.77 ± 2.93% (8 μmol/L) (Fig. 3A).Compared to the control group, as the drug concentration increased, the apoptotic proportion gradually increased (P < 0.01, P < 0.05).These results suggest that both 7a and 7j significantly promote apoptosis in the H1299 lung cancer cell line and exhibit concentration-dependent effects.www.nature.com/scientificreports/ In order to further verify the apoptotic activity induced by 7a and 7j in lung cancer cells, H1299 cells were treated with different concentrations of compounds 7a and 7j for 48 h, followed by DAPI staining.The cells were observed under a fluorescence microscope, and representative images are shown in Fig. 3B.Compared to the normal control group, H1299 cells treated with compounds 7a and 7j exhibited typical apoptotic features.As the drug concentration increased, nuclear staining intensified, and even nuclear condensation and fragmentation phenomena were observed.Therefore, the apoptosis-related results indicate that both 7a and 7j can significantly promote apoptosis in human lung cancer cells (H1299) in a concentration-dependent manner.
Transwell chamber migration assay is another important method to assess cell migration ability.Tumor cells are seeded in the chamber, and the number of tumor cells that pass through the membrane is counted to evaluate the migration ability of tumor cells 23 .The results of the Transwell migration assay (shown in Fig. 4B) also indicate that compared to the normal control group, the number of cells passing through the membrane to the lower surface of the chamber in the compound-treated group significantly decreased (p < 0.01).Therefore, the experiments demonstrate that compounds 7a and 7j can inhibit the migration ability of non-small cell lung cancer cells in a concentration-dependent manner.

Compounds 7a and 7j inhibit the clonogenic ability in H1299 cells
The colony formation assay is an important method for evaluating the ability of adherent cells to proliferate and form colonies on a plate, and it is widely used to assess cell proliferation 24 .We also employed the colony formation assay to further confirm that compounds 7a and 7j can inhibit the proliferation of lung cancer cells, as shown in Fig. 5.After treating lung cancer cells with low, medium, and high concentrations (1, 2, 4 μmol/L) of compounds for 7 days, the number of cell colonies was counted to analyze the proliferation inhibition rate.The results showed a significant decrease in the number of cell colonies in the compound-treated groups, and this effect was concentration-dependent.Even at the lowest concentration of 1 μmol/L, the compounds began to markedly inhibit the formation of H1299 cell colonies.Particularly at a concentration of 4 μmol/L, colony formation was almost completely suppressed, indicating that compounds 7a and 7j exert a strong anti-proliferative effect on H1299 cells (Fig. 5).

Compounds 7a and 7j trigger apoptosis via the apoptosis signaling pathway
We conducted an in-depth investigation into the mechanism of apoptosis induced by compounds 7a and 7j.The Bcl-2 protein family is located upstream of the apoptosis signaling pathway.Bcl-2 proteins play an anti-apoptotic role by inhibiting the release of cytochrome C (cyt-c) from mitochondria.Previous literature reports that many anticancer drugs induce apoptosis by downregulating Bcl-2 expression 25 .During apoptosis, the expression of Bcl-2 is inhibited, cyt-c is released, and it binds with the apoptotic protease activation factor to form the apoptosome.This triggers a cascade reaction of Caspase family proteins, with apoptosome promoting the activation of Caspase9.Activated Caspase9 then activates Caspase3, forming the Caspase3 cleavage body, which further activates downstream PARP protein, ultimately promoting cell apoptosis 26 .Therefore, by detecting the expression levels of Bcl-2, caspase9, caspase3, and PARP proteins, we found that the treatment with compounds 7a and 7j resulted in a decrease in Bcl-2 and caspase9 protein expression levels, while the expression levels of Cleaved-Caspase3 and Cleaved-PARP proteins increased.These results suggest that compounds 7a and 7j may effectively induce apoptosis in non-small cell lung cancer cells through the Bcl-2/caspase3/PARP signaling pathway.After treating H1299 cells with different concentrations of compounds for 48 h, cell proteins were extracted, and western blot was used to detect the expression changes of apoptosis-related proteins.The results showed that the migration-related protein MMP9 did not show significant changes in the low drug concentration group but decreased significantly in the high drug concentration group.The apoptosis-related protein Bcl-2 exhibited a decrease in expression with increasing drug concentration, while Cleaved-PARP showed an increase in expression with increasing drug concentration.The expression of caspase3, PARP, and caspase9 proteins decreased with increasing drug concentration, indicating that apoptosis in the high drug concentration group was significantly stronger than that in the low drug concentration group and the control group (p < 0.01, p < 0.05).
Previous studies have reported that MMP9 plays a crucial role in angiogenesis and cell migration 27 .In nonsmall cell lung cancer tissues, the protein expression level of MMP9 is significantly higher than in normal adjacent tissues, suggesting a close association between the high expression of MMP9 and the malignant metastasis of lung cancer as well as poor prognosis 28 .To further investigate whether compounds regulate the migration ability of non-small cell lung cancer by modulating MMP9 protein levels, Western Blot results show a decrease in MMP9 protein expression with increasing drug concentration.Therefore, compounds 7a and 7j may inhibit the migration ability of non-small cell lung cancer by downregulating MMP9 protein expression (Fig. 6).www.nature.com/scientificreports/or mitochondria 29 .BUN and CRE are typically used to reflect renal function, and an increase in their levels in the serum suggests a decline in kidney function or kidney injury 23 .The results of these biochemical indicators showed no statistical differences compared to the control group and were within the normal range.HE staining of the mouse brain, heart, liver, spleen, lungs, kidneys, and stomach further confirmed that mice did not exhibit obvious toxic characteristics at the drug concentration tested.This additional evidence indicates a high level of safety for the compounds, providing experimental data reference for the clinical safety of the compounds (p > 0.05) as shown in Fig. 7.
From the day of self-administration until the 12th day, mice in all groups showed no apparent toxic reactions, and there were no deaths.When compared to the control group, there were no significant differences in body weight among the experimental groups.(p > 0.05) as shown in Fig. 7.

Conclusion
In summary, gefitinib derivatives containing a 1,2,3-triazole ring were designed, synthesized, and evaluated for their anti-tumor activity against wild-type lung cancer cells.The synthetic method was simple and efficient, and the compounds were structurally characterized and confirmed.Several of these compounds exhibited superior antitumor activity compared to gefitinib against one or more cancer cell lines employed in this study.Among them, compounds 7a and 7j exhibited strong anti-proliferative activity in three non-small cell lung cancer.Through cell function and mechanism studies, it was found that compounds 7a and 7j downregulated the expression of Bcl-2, Caspase9, and MMP9 proteins, upregulated the expression of Cleaved-Caspase3 and Cleaved-PARP proteins, inhibited tumor cell proliferation and migration, promoted cell apoptosis.Acute toxicity experiments in mice also confirmed their safety, providing foundational data for future in vivo experiments and clinical applications of these novel gefitinib derivatives, with the hope that these compounds may become effective and low-toxicity anti-cancer drugs.

Materials and Chemistry
The gefitinib-1,2,3-triazole derivative was synthesized in-house.All the reagents and solvents used were obtained from a commercially available source.The 1 H and 13 C NMR spectra were acquired in a DMSO-d 6 solution using a Bruker 400 MHz NMR spectrometer.High-resolution mass spectra (HRMS) measurements were carried out using a Bruker Compact mass spectrometer.

General procedure for the preparation of compound 7
Aryl-azido (150 mg, 0.7 mmol) and 6 (250 mg, 0.56 mmol) were added to 15 mL mixed solvent (water/tertbutanol/THF = 1:1:1).Copper sulfate pentahydrate (14 mg, 0.1 mmol) and sodium ascorbate (23 mg, 0.1 mmol) were added and the mixture was stirred at 85 °C for 12 h.After the completion of the reaction (monitored by TLC), the mixture was extracted with dichloromethane (15 mL × 3).The combined organic phase was washed successively with brine, dried over sodium sulfate and concentrated in vacuo.The residue was purified by through column chromatography (CH 2 Cl 2 /MeOH = 20:1) to give the desired compound 7a-7t as a crystalline powder.

CCK-8 assay for cell proliferation and cytotoxicity
Three types of lung cells were seeded into 96-well plate at a density of 5 × 10 3 cells per well at logarithmic growth phase and cultured in 37 °C.Then, the cells were treated initially with different concentrations of compounds (0, 2, 4, 8, 16 and 32 μmol/L) for additional 48 h, with three replica wells each.After that, cell viability was determined according to the instruction of the CCK-8 assay.Next, 10 μL of CCK-8 solution was added to each well of the plate and incubate the plate for 1-4 h in the incubator.The absorbance was measured at 450 nm using a microplate reader (Bio-Tek).The percentage of viable cells was measured using the following formula where three independent experiments were performed: [(A450 sample − A450 blank )/(A450 control − A450 blank )] × 100%.
Flow cytometry detection for cell apoptosis H1299 cells were seeded in a 6-well plate at a density of 1 × 10 5 cells/well and incubated for 24 h.On the following day, the medium was replaced with fresh medium containing 7a or 7j (2, 4 and 8 μmol/L) and cells were incubated for an additional 48 h.Cell apoptosis was detected using Annexin V-EGFP/PI apoptosis detection Kit by flow cytometry.Subsequently, 5 µl of EGFR Annexin V and 10 µl of PI was added to the cell suspension, gently vortexed, and incubated at room temperature in the dark for 20 min, then single-cell suspension was prepared followed by flow cytometry (BD Accuri™ C6 Plus).
DAPI staining for cell apoptosis H1299 (1.5 × 10 4 ~ 2 × 10 4 /well) cells were seeded in 24-well plates for 24 h, and then treated with 7a or 7j at the concentrations of 2,4 and 8 μmol/L for 48 h., the cells were fixed for 30 min at room temperature in 4% paraformaldehyde and washed 3 times in PBS.Finally, every well was stained with DAPI (10 µg/mL, C0065, Solarbio) at room temperature for 30 min, and washed at least three times in PBS.The fluorescence microscope was used to observe the morphological changes of cell nuclei.
Wound healing assay H1299 cells were seeded in a 6-well plate, When the cell confluency reached 95%, a wound line was scratched using a 200 µl pipette tip, and then washed three times, fresh medium containing 7a or 7j (2, 4 and 8 μmol/L) was added, and the plate was incubated at 37 °C with 5% CO 2 .All the images were captured at 0 h and 24 h under an inverted microscope, and the quantification of scratches was analysed using Image J.

Transwell migration assay
Transwell assay was used to determine the migration ability of H1299 cells.Cells were starved in serum-free RPMI 1640 medium for 24 h, then detached and resuspended in serum-free RPMI 1640 medium.5 × 10 5 cells/ mL were inoculated into the upper chamber of a 24-well Transwell plate (Corning Inc., United States), with a volume of 100 µl per well.Then, 100 µL serum-free medium containing 7a or 7j (2, 4 and 8 μmol/L) was added to the upper chamber and the lower chamber was filled with 700 µL medium containing 20% FBS.After incubation for 24 h, H1299 cells on the upper membrane of the transwell were wiped off.The migrated cells were treated with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 20 min and washed three times with PBS.The number of H1299 cells that migrated to the underside of the membrane was counted under the inverted fluorescence microscope.Three randomly selected areas from each transwell were photographed and calculated using Image J.
Colony formation assay H1299 cells were seeded in six-well plates at a density of 500 cells per well and treated with 7a or 7j (1, 2 and 4 μmol/L) for 7 days.Then, cells were per-fixed with 4% paraformaldehyde for 30 min, stained with 0.1% Crystal violet for 15 min, and then washed with pure water.Taking a picture after the plates were air-dry.The clone formations number was counted with Image J.

Western blot analysis
Firstly, H1299 cells were treated with different concentrations concentrations of 7a and 7j at 4, 8 and 16 μmol/L for 48 h, cells were harvested using RIPA lysate (R0010, Solarbio) and centrifuged at 12,000 rpm for 15 min at 4 °C, and then the concentration of total protein was measured and taken from the supernatant.Protein was separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membrane.after blocking in 5% milk for 2 h, the membranes were incubated with specific primary antibody at 4 °C overnight.
Next day, NC membranes were incubated for 1 h at room temperature with appropriate secondary antibodies, and then Protein bands was visualizing by chemiluminescence detection (ECL kit, Genview).

Figure 3 .
Figure 3. Cell apoptosis induced by 7a and 7j in H1299 cells.(A) Apoptosis quantification detected by Annexin V-EGFP/PI staining.(B) Cell apoptosis morphological changes detected by DAPI staining.Data were mean ± SD. n = 3 for each concentration.
https://doi.org/10.1038/s41598-024-60000-1www.nature.com/scientificreports/Acuteoral toxicity assessmentWe further evaluated the safety of the compounds through acute toxicity experiments in mice.From the initial gavage to the 12th day, there was no statistical difference in mouse body weight compared to the control group.Observation of organ morphology during dissection revealed no pathological changes, and organ indices showed no statistical differences compared to the control group.Biochemical indicators, including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE), were measured.ALT and AST are commonly used to assess liver function, reflecting physiological and pathological changes in liver function.Elevated levels of ALT and AST in the serum indicate damage to liver cells

Figure 4 .Figure 5 .
Figure 4. Cell migration and invasion inhibition induced by 7a and 7j in H1299 cells.(A) Photographs of cells at 0 and 24 h after treatment with different concentrations of drugs (scale bar = 100 μm).(B) Cell migration and invasion inhibition induced by 7a and 7j in H1299 cells.

Figure 6 .
Figure 6.Assessment of apoptosis and migration induced by 7a and 7j.(A) Western blotting results of the protein levels in H1299 cells treated with 4, 8 or 16 μM.compounds 7a.Numbers below each lane indicated the relative expression level of the protein.(B) Western blotting results of the protein levels in H1299 cells treated with 4, 8 or 16 μM.compounds 7j.Numbers below each lane indicated the relative expression level of the protein.

Figure 7 .
Figure 7. Compound 7a in vivo toxicity of mice.(A) Acute toxicity experiments of compound 7a were conducted in mice.Mouse body weight changed.(B) The acute toxicity experiments examined the effects of compound 7a on mouse organs (C) Effect of acute toxicity experimental studies on blood biochemical indices in mice.(D) H&E staining were performed on various organs of mice treated with compound 7a.